Antibiotics em-2,em-3 and em-4 and process for producing same

ABSTRACT

New antibiotics, EM-2 (U-40588), EM-3 (U-40589), and EM-4 (U40590), produced by the controlled fermentation of a strain of the known microorganism Emericellopsis microspora in an aqueous nutrient medium to which propylproline is added. These antibiotics are primarily active against Gram-positive bacteria and can be used in various environments to eradicate or control such bacteria.

United States Patent Argoudelis et al.

[11] 3,821,367 [4 June 28, 1974 ANTIBIOTICS EM-2, EM-3 AND EM-4 AND PROCESS FOR PRODUCING SAME Inventors: Alexander D. Argoudelis, Portage;

LeRoy E. Johnson, Kalamazoo Twp., Kalamazoo County, both of Mich.

The Upjohn Company, Kalamazoo, Mich.

Filed: Oct. 24, 1972 Appl. No.: 300,433

Assignee:

U.S. Cl 424/119, 424/120, 195/80 Int. Cl A6lk 21/00 Field of Search 424/119, 120; 195/80 References Cited UNITED STATES PATENTS 12/1971 Higashide et al. 424/120 3,697,649 10/1972 Reimann 424/120 Primary Examiner-Jerome D. Goldberg Attorney, Agent, or Firm-Roman Saliwanchik dium to which propylproline is added. These antibiotics are primarily active against Gram-positive bacteria and can be used in various environments to eradicate or control such bacteria.

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ANTIBIOTICS EM-2, EM-3 AND EM-4 AND PROCESS FOR PRODUCING SAME BRIEF SUMMARY OF THE INVENTION The novel compounds of the invention, EM-2, EM-3, and EM-4 are obtained by culturing Emericellopsis microspora strain 333, NRRL 5648, in an aqueous nutrient medium to which propylproline, e.g., 4-trans-n-propyl-L-proline is added. These compounds are neutral compounds and have the property of adversely affecting the growth of Gram-positive bacteria, for example, Staphylococcus aureus, Streptococcus hemolyticus, Streptococcus faecalis, Bacillus subtilis', Diplococcus pneumoniae, and Sarcina lutea; they also demonstrate some Gram-negative activity and antiprotozoan activity. Accordingly, these antibiotics can be used alone or in combinationwith other antibiotic agents to prevent the growth of or reduce the number of bacteria, as disclosed above, in various environments.

DETAILED DESCRIPTION OF THE INVENTION CHEMICAL AND PHYSICAL PROPERTIES OF I. Elemental Analysis:

C, 57.29; H, 7.66; N, 13.22; 0, 21.83.

Found: C, 57.47; H, 7.70; N, 12.99; 0, 21,87 (by difference). 2. Melting Point: 26l.4 C. 3. Specific Rotation: [01],, +5 (c, 1.0, methanol) 4. Ultraviolet Absorption Spectrum:

In methanol:

A max (my) a 216(sh) 24.02 269(sl.sh) 2.67 273 2.81 l

- analysis and the hydrolysate was analyzed for aminoacid content. Results are presented in Table A, infra. 8. Infrared Absorption Spectra:

Infrared absorption spectrum of antibiotic EM-Z suspended in a mineral oil mull is reproduced in FIG. 1 of the drawing. Antibiotic EM-Z shows peaks at the following wave lengths expressed in reciprocal centimeters:

Band Frequency Band Frequency (cm") Intensity (cm") Intensity 3400 S 2860 (oil) S 3292 S 1650 S 3060 W 1636 S 2960 (oil) S 1538 S 2928 (oil) 5 1530 S 1455 S 1166 M I415 S 1145 M 1410 M I075 M I378 M 1006 M 1375 M 967 W 1360 M 922 W I337 M 867 \V 1300 M 800 \V 1280 M 740 M 1225 M 700 M I200 M Following is a tabulation of the infrared absorption spectrum wave lengths where the compound is pressed into a KBr pellet. 1

Band Frequency Band Frequency (cm") Intensity (cm") Intensity 3420 S 1362 M 3320 S 1337 M 3060 W 1282 M 2965 M I M 2935 M 1147 M 2875 M I075 M I645 S 1035 W 1538 S 1015 W I530 S 965 W I464 M 873 W I455 M 800 W I408 M 740 M 1380 M 700 M Infrared band intensities, throughout this disclosure, are indicated as S, M, and W'," respectively, and areapproximated in terms of the backgrounds in the vicinity of the bands. An S band is of the same order of intensity as the strongest in the spectrum; M" bands are between one-third and two-thirds as intense as the strongest band; and, W bands are less than one-third as intense as the strongest band. These estimates are made on the basis of a percent transmission scale. sh denotes a shoulder.

CHEMICAL AND PHYSICAL PROPERTIES OF EM-3 l. Elemental Analysis:

CalCd for CgaHumNwOmi C, 57.83; H, 7.73; N, 12.89; 0, 21.55.

Found: C, 58.46; H, 7.78; N, 13.27; 0, 21.49 (by difference). 2. Melting Point: 256.9 C. 3. Specific Rotation: [011 +12 (0, 1.0, methanol) 4. Ultraviolet Absorption Spectrum:

In methanol:

A max (mu) a 243(sl.sh) 0.48 253 0.43 257 0.46 261 (sh) 0.43 264 0.44 267 0.41

5. Potentiometric Titration:

No titratable group was found when an EM-3 preparation was titrated in 72 percent aqueous ethanol with either sodium hydroxide or hydrochloric acid as titrants. 6. Solubilities:

Antibiotic EM-3 has the same solubility characteristics as antibiotic EM-2, supra. 7. Aminoacid Composition:

A preparation of antibiotic EM-3 was analyzed for aminoacid composition as disclosed above for antibiotic EM-2. Results are presented in Table A, infra.

8. Infrared Absorption Spectra:

Infrared absorption spectrum of antibiotic EM-3 suspended in a mineral oil mull is reproduced in FIG. 2 of the drawing. Antibiotic EM-3 shows peaks at the following wave lengths expressed in reciprocal centimeters:

Band Frequency Band Frequency (cm Intensity (cm Intensity 3430 M I380 M 3395 M I373 M 3325 S 1360 M 33I5 S I324 M 3060 W I3I0 M 2960 (oil) S 1285 M 2920 (oil) S I273 M 2855 (oil) S I226 M I680 S I217 M l65l S I205 M I622 S I178 M l6l 3 S I165 M I537 S ll30 W I530 S I072 W I460 S I047 W I422 M 1035 W I018 W 840 W I010 W 798 W 992 W 775 W 967 W 755 W 925 W 723 M 910 W 703 M 870 W 695 M 857 W Following is a tabulation of the infrared absorption spectrum wavelengths where the compound is pressed into a KBr pellet.

Band Frequency Band Frequency CHEMICAL AND PHYSICAL PROPERTIES OF EM-4 1. Elemental Analysis:

Calcd for Cg1H121 l7 22 C, 57.55; H, 7.52; N, 14.09; 0, 20.84. Found: C, 57.26; H, 7.45; N. 14.22; 0. 21.08 (by difference). 2. Melting Point: 239.8 C. 3. Specific Rotation: [01],, =+l3.5 (c, 1.0, methanol) 4. Ultraviolet Absorption Spectrum:

In methanol:

A max (mg) a 24mm 0.26 252 0.24 257 0.26 26I(sh) 264 0.26 2 7 0.15

5. Solubilities:

Antibiotic EM-4 has the same solubility characteristics as antibiotic EM-Z, supra. 6. Aminoacid Composition:

A- preparation of antibiotic EM-4 was analyzed for aminoacid composition as disclosed above for antibiotic EM-Z. Results are presented in Table A, infra.

7. Infrared Absorption Spectra:

Infrared absorption spectrum of antibiotic EM-4 suspended in a mineral oil mull is reproduced in FIG. 3 of the drawing. Antibiotic EM-4 shows peaks at the following wave length expressed in reciprocal centimeters:

Band Frequency Band Frequency Following is a tabulation of the infrared absorption spectrum wave lengths where the compound is pressed into a KBr pellet.

Band Frequency Band Frequency (cm") Intensity (cm") Intensity 3400(Sh) S 3060 W 3320 S 3020 W 2980 M I361 M 2960 M I330 W 2930 M I230 M 5 2870 W 1220 M I648 S ll M I620 5 I077 W I558 M I028 W I533 S 970 W -Continued Band Frequency Band Frequency Following Tables A and B are additional characterizations of the antibiotics of the subject invention. Table A gives the aminoacid composition of the antibiotics and also compares them with the well-known antibiotic antiamoebin. Table B gives the antibacterial spectrum of the antibiotics of the subject invention and compares them with the antibiotic antiamoebin. This antibacterial spectrum was determined in a standard two-fold broth dilution test in Brain Heart Infusion broth wherein a final dilution of a fully grown culture in Brain Heart Infusion of l/40,000 is made. Test organisms requiring blood are cultured in this, but the final antibiotic test is made in the absence of blood, since it has been found that adequate growth is obtained without it. Incubation is unshaken at 37 C. End points are read at 20 hours. Brain Heart Infusion broth (supplied by Difco Laboratories, Detroit, Mich., United States of America) has the following composition:

Calf brains, infusion from 200 gm. Beef heart, infusion from 250 gm. Bacto proteose-peptone, Difco gm. Bacto-Dextrose. Difco 1 gm. Sodium chloride 5 gm. Disodium phosphate 2.5 gm. Water I 1000 ml.

TABLE A The antibiotics of the subject invention also show antiprotozoa] activity. The growth inhibition of the protozoa (O. danica and C. fasciculata) was determined by two-fold dilution end points in a medium consisting of:

Glucose l0gm/l Bacto Proteosepeptone IOgm/l No. 3 (Difco) Yeastolate (Difco) lgm/l The protozoa were grown at 26 C. for 48 hours, diluted to a final concentration of 1:1,000; and the dilution' tubes then incubated at 26 C. for 48 hours and end points read. The results are as follows:

Protozoa EM-2 EM- 3 EM-4 Ochromonas 50mcg/ml" 200mcg1ml 200mcg/ml danica Crirhidia SOmcg/ml" 200mcg/ml' 200mcg/ml fasciculata Terrahymena SOmcg/ml 200mcg/ml 200mcg/ml pyrtfarmir Concentration which inhibited the growth of the protozoa. "Concentration which partially inhibited the growth of the protozoa.

Aminoacid Composition of EM-Z, EM-3, EM-4 and Antiamoebin EM-2 EM-3 EM-4 Antiamoebin moles pmolcs pirnoles moles Aminoacid Wt per mg Wt (92) per mg Wt (76) per mg Wt per mg Lysine 3.18 0.221 Hydroxypmline 12.53 0.972 16.92 1.220 15.24 1.230 12.07 0.920 Thrconinc 5.01 0.427 Glutamic 13.39 0.925 8.65 0.555 8.32 0.598 7.02 0.477 Froline 6.67 0.589 6.03 1 0.524 Glycine 5.11 0.643 4.55 0.640 3.69 0.491 Alanine 4.43 0.470 0.43 0.051 1.46 0.164 a-Aminoiso' butyric acid 3.44- 0.346 2.91 0.272 5.84 0.611 4.82 0.477 Valinc 1.78 0.154 7.47 0.602 7.08 0.639 1.97 0.168 lsolcucine 1 1.60 0.899 Lcucinc 7.02 0.544 8.03 0.578 7.68 0.619 7.27 0.544 Phenylalanine 9.64 0.551 10.03 0.642 8.64 0.523

TABLE B tions 666; both antiamoebinand stilbellin and have I v been separated from antiamoebin and stilbellin by thin ANTIBACTERIAL SPECTRUM 0F ANTIBIOTICS layer chromatography (tlc) and paper chromatogra- AND ANT'AMOEB'N phy. Drawings of the above comparisons of the antibi- Minimum Inhibitory Concentration (meg/ml) EM-2 EM-3 EM-4 Antiamoebin Test Organism meg/ml meg/m1 mcg/ml meg/ml S. aureus UC 76 4 31 31 62 S. aureus 552 8 3! 62 125 S. hemolyticus I52 2 16 31 62 S. faecalir 3235 8 62 62 125 E. coli 5] 500 250 500. 500 P. vulgaris 93 500 250 500 500 K. pneumoniae 57 500 250 500 500 S. srhorrmuelleri 126 500 250 500 500 P8. aeruginosa 95 500 250 500 500 B. rubnlis 564 8 31 3| 31 D. pneumoniae 41" 2 31 62 .r. lulea 2 8 16 3| Requires blood for growth Note: The prefix "UC" refers to The Upjohn Company culture collection.

solvent system. Twenty microliters of antibiotic sample was applied to the sheets. The sheets were developed for approximately two hours, dried, and then applied to Sarcina Iutea trays. S. lutea is inoculated into the assay agar (Seed Agar Med. No. l, BBL, Cockeysville, Md.) of the following composition:

Gelysate Peptone 6.0gm/l Trypticase Peptone 4.0gm/l Yeast Extract 3.0gm/l Beef Extract 1.5gm/l Glucose 1.0gm/l Agar l gm/l pH 6.6102

The trays are incubated at 28 C. for 18 hours and the zones observed by llc are recorded as to R, value.

THE MICROORGANISM The microorganism used for the production of the antibiotics of the subject invention is a strain of the known microorganism Emericellopsis microspora. One of its strain characteristics is the production of antibiotics EM-2, EM-3 and EM-4 in the presence of propylproline. A subculture of the living organism was deposited and can be obtained from the permanent collection of the Northern Utilization and Research Service, U.S. Department of Agriculture, Peoria, lll., U.S.A. lts accession number in this repository is NRRL 5648.

The microorganism of this invention was studied and characterized by Alma Dietz of the Upjohn Research Laboratories.

Emericellopsis microspora strain 333 has the macroscopic and microscopic growth characteristics of the known microorganism Emericellopsis microspora WSF-4'7, ATCC 14645, as described by Backus and Orpurt [Backus, MR and PA. Orpurt. 1961. A new Emercellopsis from Wisconsin with notes on other species. Mycologia 53:64-83] Emercellopsis microspora strain 333 is distinguishable from Emercellopsis microspora WSF-47 by the fact that Emercellopsis microspora strain 333 requires the presence of propylproline in the fermentation medium to produce the antibiotics of the subject invention.

The new compounds of the invention are produced when the elaborating organism is grown in an aqueous nutrient medium under submerged aerobic conditions in the presence of propylproline. It is to be understood also that for the preparation of limited amounts surface cultures and bottles can be employed. The organism is grown in a nutrient medium containing a carbon source, for example, an assimilable carbohydrate, and a nitrogen source, for example, an assimilable nitrogen compound or proteinaceous material. Preferred carbon sources include glucose, brown sugar, sucrose, glycerol, starch, cornstarch, lactose, dextrin, molasses, and the like. Preferred nitrogen sources include cornsteep liquor, yeast, autolyzed brewer's yeast with milk solids, soybean meal, cottonseed meal, cornmeal, milk solids, pancreatic digest of casein, distillers solids, animal peptone liquors, meat and bone scraps, and the like. Combinations of these carbon and nitrogen sources can be used advantageously. Trace metals, for example, zinc, magnesium, manganese, cobalt, iron, and the like, need not be added to the fermentation media since tap water and unpurified ingredients are used as components of the medium.

The antibiotics of the subject invention are produced when an effective amount of propylproline, e.g. 4-trans-n-propyl-L-proline is added to the Emericellopsis microspora strain 333 fermentation. The effective amount of propylproline can be varied from about 1 mg./l. to about 4 gm./l. High levels detract from the economic advantages of the process. It has been found that the effective amount of propylproline will vary with media ingredients employed, and that high antibiotic fermentation titers are produced at levels of about 1 to 2 gm./l. The propylproline can be added to the fermentation, conveniently, at any time after inoculation, or at intervals during the course of the fermentation. The preferred propylproline of the subject invention is 4(R)-propylproline. A mixture of 4(R) and 4(8) propylproline can be used if the mixture contains 50 percent or more of the 4(R) configuration. Propylproline can be prepared as disclosed in pending U.S. application Ser. No. 220,389, filed Jan. 24, 1972.

Production of the compounds of the invention can be effected at any-temperature conducive to satisfactory growth of the microorganism, for example, between about 18 and 40 C., and preferably between about 20 and 32 C. Ordinarily, optimum production of the compounds is obtained in about 2 to 10 days. The medium normally remains basic during the fermentation. The final pH is dependent, in part, on the buffers present, if any, and in part on the initial pH of the culture medium.

When growth is carried out in large vessels and tanks, it is preferable to use the vegetative form, rather than the spore form, of the microorganism for inoculation to avoid a pronounced lag in the production of the new compounds and the attendant inefficient utilization of the equipment. Accordingly, it is desirable to produce a vegetative inoculum in a nutrient broth culture by inoculating this broth culture with an aliquot from a soil or a slant culture. When a young, active vegetative inoculum has thus been secured, it is transferred aseptically to large vessels or tanks. The medium in which the vegetative inoculum is produced can be the same as, or different from, that utilized for the production of the new compounds, as long as it is such that a good growth of the microorganism is obtained.

A variety of procedures can be employed in the isolation and purification of the compounds of the subject invention, for example, solvent extraction, partition chromatography, silica gel chromatography, liquidliquid distribution in a Craig apparatus, absorption on resins, and crystallization from solvents.

In a preferred recovery process, the compounds of the invention are recovered from the culture medium by separation of the mycelia and undissolved solids by conventional means, such as by filtration or centrifugation. The antibiotics are then removed from the filtered or centrifuged broth by resin sorption on a resin comprising a non-ionic macro porous copolymer of styrene crosslinked with divinylbenzene. Suitable resins are Amberlite XAD-l and XAD-2 disclosed in U.S. Pat.

No. 3,515,717. After the resin is washed with water, it

is eluted with an organic or aqueous organic solvent in which the sorbed antibiotic is soluble. Ninety-five percent aqueous methanol is the preferred solvent for elution. Fractions from the column are tested for bioactivity on a standard microbiological test using the microorganism Sarcina lutea. Active fractions are subjected to further recovery procedures, for example, solvent extraction. In a preferred process, active fractions from the resin column are mixed with acetone and the remaining filtrate is concentrated to dryness. The residue obtained is dissolved in methanol and this solution is mixed with ether to give a precipitate containing the antibiotics of the subject invention. Separation of the 9 antibiotics is achieved by silica gel chromatography using silica gel (MerckDarmstadt Art 7734) and the solvent system chloroform-methanol (6:1 v/v).

Some antibiotic activity is also present in the spent beer from the resin column, described above. This antibiotic activity is recovered from the spent beer and resolved into the individual antibiotics by butanol extraction of the spent beer and silica gel chromatography of the butanol extracts.

Another source of antibiotics from the fermentation disclosed herein is the filter cake from the filtration operation. In a preferred process, the filter cake is triturated with methanol and the methanol extracts are then subjected to silica gel chromatography, as described above, to give the individual antibiotics in their pure form. r

The antibiotics of the invention are active against Staphylococcus aureus and Streptococcus foecalis and can be used to disinfect washed and stacked food utensils contaminated with these bacteria; they can also be used as disinfectants on various dental and medical equipment contaminated with Staphylococcus aureus. Further, since the antibiotics of the invention are active against Streptococcus hemolyticus, they can be used to disinfect instruments, utensils, or surfaces, where the inactivation of this microorganism is desirable.

It is to be understood that the microbiological process disclosed herein, though described in detail with reference to the strain of Emericellopsis microspora strain 333, NRRL 5648, is not limited to this particular microorganism or to microorganisms fully described by the cultural characteristics disclosed herein. It is intended that this invention also include other strains or mutants of the said microorganism which can be produced by procedures well known in the art, for example, by subjecting the novel microorganism to x-ray or ultra-violet radiation, nitrogen mustard, phage exposure, and the like.

Hereinafter are described non-limiting examples of the process and products of the subject invention. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

EXAMPLE 1 Part A. Fermentation A soil stock of Emericellopsis microspora strain 333 NRRL 5648, is used to inoculate a series of SOD-ml. Erlenmeyer flasks, each containing 100 ml. of sterile seed medium consisting of the following ingredients:

Glucose monohydrate 10 gm/i Bacto Peptone (Diico) i0 gmli Bacto Yeast Extract (Difco) 2.53m" Deionised water. pH is Balance mentation medium.

The fermentation medium consists of the following ingradients:

Glucose monohydrate 455m" Buffalo starch 40|mli Biackstrnp molasses 203ml] Calcium carbonate Blm/i Potassium sulfate 2 m/i Peptone (Wilson's s.p.

granular" 25|mli Tap water q.|. Balance A corn starch supplied by the Corn Products Corporation international.

llilno s.

"Su piled by the Wilson Pharmaceutical and Chemical Corporation, f.hica o.

vious preparations, are as follows:

The pH of the fermentation medium is adjusted to 7.2 with an aqueous solution of sodium hydroxide before sterilization. Three drops of Ucon (a synthetic defoamer supplied by Union Carbide, N.Y., NY.) is added per flask. The inoculated fermentation flasks are incubated for 24 hours at a temperature of 28 C. on a Gump rotary shaker operating at 250 rpm. After 24 hours, propylproiine is added to the flasks (50 mg. per 100 ml. of medium). The flasks are then reincubated on the Gump rotary shaker. Harvest is usually after 192 hours of fermentation.

The total antibiotic titer of the fermentation beer can be monitored by use of a disc plate assay using the microorganism S. lutea, described above.

Part B. Recovery Whole fermentation beer (10 liters), harvested 168 hours after the addition of propylproiine, obtained as described above, is filtered using diatomaceous earth as a filter aid. The filter cake is washed with 1 liter of water. Clear beerand the water wash arecombined (8 1iters) and kept as preparation 101.1. The filter cake is triturated. with 2 liters of absolute methanol to give a methanolic extract and kept as preparation 101.2.

Preparation 101.1 is passed over a, chromatography column containing 500ml. of Amberiite XAD-2. The spent beer is collected as one fraction and designated as preparation 101.3. The column is then washed with 2 liters of water. The aqueous wash; is kept as preparation 101.4. The column is eluted with percent aqueous methanol. Fractions of 20 m1. are collected and selected fractions are tested for bioactivity against S. lutea. The results of these tests, as well as tests on the pre- S. iutea zone (mm) Preparation I 101.1 Clear beer 21 10 1.2 'Methanolic extract 12 101.3 Spent beer from XAD-2 chromatography 18 101.4 Aqueous wash from XAD-2 chromatography 9 Fractions eluted with 95 percent methanol:

S. Iutea zone (mm) Fractions 14-23 are combined and concentrated to dryness to give preparation 102.1. This preparation is processed further in Part C (.4). Part C. Purification 1. isolation of the Antibiotic Activities Present in Spent Beer by Extraction with Butanoi Preparation 101.3 (8iiters), obtained as described in Part B, is extracted twice with 2.5 liters of i-butanol each time. The butanol extracts are combined and con centrated todryness to give preparation 141.3.

2. isolation of the Antibiotics Present in the Buta-.

noiic Extract of Spent Beer by Silica Gel Chromatogra- P lii...

Preparation 141.3, obtained as described in Part C (1 is triturated with 250 ml. of acetone. The resulting insoluble material is isolated by filtration and dried; yield, 6.4 gm. of preparation 141.4. This preparation is found by tlc to be a mixture of antibiotics EM-3 and EM-4 and is purified by silica gel chromatography as described below.

The chromatography column is prepared from 1 kg. of silica gel packed in the solvent system consisting of chloroform-methanol (6:1 v/v). The starting material for the chromatography, preparation 141.4 (6 gm.), is dissolved in 300 ml. of methanol. This solution is mixed with 600 ml. of chloroform and 50 gm. of silica gel. The mixture is concentrated to dryness and the dry powder obtained is added on the top of the column. The column is eluted with the above solvent system. Fractions (20 ml.) are collected and tested for bioactivity against S. lutea. Selected fractions are analyzed for antibiotic content by :10 chromatography.

Fractions 1-269 are found to be inactive.

Fractions 270-500 are found to contain antibiotic EM-4 only. These fractions are combined and the solution is concentrated to dryness to give 1.2 gm. of colorless crystalline material designated preparation 57.1. This preparation is recrystallized from 36 ml. of 50 percent aqueous methanol; yield, 900 mg. of colorless crystalline needles of antibiotic EM-4.

Fractions 501-600 also contain the antibiotic EM-4 only. These fractions are combined and the slightly colored solution is concentrated to dryness to give 210 gm. of preparation 56.2.

Fractions 601-1165 contain both antibiotics EM-3 and EM-4. These fractions are combined and the solution is concentrated to dryness to give preparations designated 56.3 and 56.4. Antibiotic EM-3 is obtained from these preparations and others as described in Part C (7).

3. Isolation of the Antibiotics Present in the Methanolic Extract of the Filter Cake Preparation 101.2 (2 liters), obtained as described in Part B, is concentrated to dryness. The resulting residue is triturated with 1 liter of absolute methanol. The filtrate is mixed with 5 liters of acetone. The resulting precipitate is kept as preparation 1 14.2 (5.7 gm.). The new filtrate is mixed with 20 liters of ether. The new precipitate formed is isolated by filtration and dried and kept as preparation 114.3 (7.4 gm.). Preparation 1 14.3, as shown by tlc, contains antibiotics EM-2 (small amounts), EM-3 and Em-4. Separation of these antibiotics is achieved by silica gel chromatography.

The silica gel chromatography column is prepared from 1.8 kg. of silica gel packed in the solvent system consisting of chloroform-methanol (6:1 v/v). The starting material, preparation 1 14.3 (ca 7 gm.), obtained as described above, is dissolved in the above solvent system (500 ml.) and this solution is mixed with 100 gm. of silica gel. The mixture is concentrated to dryness and the obtained powder is added on the top of the column. The column is eluted with the above solvent system. A total of 2,100 fractions (20 ml. each) are collected. Then 15 fractions of 2 liters each are collected by elu-I, tion of the column with the same solvent. Selected fractions are analyzed for bioactivity against S. lutea and for antibiotic content by tlc.

Fractions 1-939 are found inactive and are discarded.

Fractions 940-1200 contain antibiotic EM-4 only. They are concentrated to dryness; yield, 1.62 gm. of colorless crystalline material designated preparation 31.1. This material is recrystallized from 48 m1. of 50 percent aqueous methanol to give 1.4 gm. of colorless needles designated preparation 107.1. This material is found by tlc to be antibiotic EM-4.

Fractions 1201-1250 also contain antibiotic EM-4. These fractions are concentrated to dryness to give preparation 31.2 (360 mg).

Fractions 1351-1950 contain both antibiotics EM-3 and EM-4. These fractions are combined and the solution is concentrated to dryness and designated preparations 31.3 and 31.4 which are treated as described in PartC (7).

Fractions (2 liters each) 6-13 contain antibiotic EM-2. These fractions are combined and concentrated to dryness to give preparation 31.5 (320 mg). This preparation is processed further as disclosed in Part C (6).

4. Isolation of the Antibiotics Present in the Amberlite XAD-2 Eluates by Silica Gel Chromatography Preparation 102.1 (160 m1. of 95 percent methanolic eluates from the Amberlite XAD-2 chromatography described above in Part B) is mixed with 1.6 liters of acetone. The resulting precipitated material is separated by filtration and discarded. The filtrate is concentrated to dryness to give a residue which is dissolved in 50 m1. of methanol, and this solution is mixed with 2 1iters of ether. The resulting precipitated material is isolated by filtration, dried, and kept as preparation 1 16.1 (2.1 gm.). This preparation, as determined by tlc. contains mainly EM-4 and EM-3. Separation of these antibiotic activities is achieved by silica gel chromatography as described below.

The chromatography column is prepared from 200 gm. of silica gel (Merck-Darmstadt Art 7734) packed in chloroformmethanol (6:1 v/v). Preparation 116.1

(2.1 gm.) is dissolved in 20 ml. of methanol and 100 ml. of chloroform. This solution is mixed with gm. of silica gel and the mixture is concentrated to dryness. The dry powder obtained is added on the top of the column. The column is eluted with the above solvent system. Fractions of 20 ml. are collected and analyzed by testing against S lutea after ten-fold concentration. Results follow:

Fraction No. Zone (mm) Fraction No. Zone (mm) 5 0 230 16 10 0 240 17.5 15 0 250 16 20 0 260 15 0 g 270 I5 0 280 14 0 290 13.5 0 300 13 0 310 12.5 0 320 12.5 0 330 10 6O 0 340 9 0 350 traces 0 360 do. 0 370 do. 0 380 do. v 0 390 do. (l 400 do. trucex 410 do. 14 421) do. 16.5 430 do. 1 10 17.5 440 do. 17.5 450 8 g 17.5 460 1 1 16.5 470 1 1 17 480 11 17 490 11 17. 500 11 16.5 510 11 16 520 ll 530 10 -New S. 20.5 540 10 1601mm tray 20.5 550 10 .16 wasu s 1 v599..

i n N n m) Fr i l l Preparation 126.2 and 126.3 are discarded.

Hg 38 :38 g 5. Crystallization of Antibiotic EM-4 180 20 590 8 Preparation 126.1 is found to contain antibiotic 185 19,5 600 0 EM-4. This preparation (500 mg.) is recrystallized {3 3:; $8 8 5 from 15 ml. of 5 percent aqueous methanol. The color- 200 19.5 630 0 less needles obtained are isolated by filtration and 3g :2 228 3 dried; yield, 410 mg. of crystalline antibiotic EM'4. v I I u WU I 6. Crystallization of Antibiotic EM-Z Preparations 137.2 and 31.5, obtained as described Fractions l-l 75 are found by tlc to contain antibiotic EM4. These fractions are combined and the soluif. ffi C C respesc 85 i tion is concentrated to dryness. The resulting crystal- 3:22 :23 l 8 :5: s f g 3 23322 2? der Ega line residue is triturated with 50 ml. of acetone. The reon a steam bath methanofic solution is clarified g g i a filtration and gried; filtration and mixed with water to a final volume of 45 yie mg. 0 anti iotic preparation esig- 1 nated 126.1. This preparation is processed further as gggg'is fixg 2:5 3:22 g fg a i g disclosed in Part C (5). The filtrate, ca 60 ml., 1s mixed 7 isolation and C stallizafionqf Anfibiofic with 600 ml. of ether. The precipitate formed is col- YE 56 4 P t C lected and designated preparation 126.2 (50 mg). The fi ga g t C new filtrate is concentrated to dryness and the resulting d an g l b o 1 residue is dissolved in 1 ml. of methanol and 1 ml. of i 36 g g v e a mixture? 0 0t acetone. This solution is mixed with 40 ml. of ether and 233 3 23 0 g j ig a zg g lg gii ig 50 m1. of Skellysolve B (isomeric hexanes). A new precipitate is collected and designated as preparation gg jggmg ggzgg'; g g s s g s fggg sri l i fi. (t2Odmg.:.S;l':1:gf v gilxgigfifig lgzi colleen-1 silica gel (Merck-Darmstadt Art 7734) packed in the ra e o ryn 1 v Fractions 176-300, from the above silica gel chroma- :ilfigfiggggg 1:23:5 $$fifi22i L9 i g i i g g g comam bothbantg described above is in 200 ml of the solvent System 1ot1cs an ese rac ions are com me and the solution is concentrated to dryness to give f i z i z preparation 137.1, which is trated as described in Part g g gg gg fi p gsi g ggg g g l r z ctions 450-590, from the above-described silica is eluted with the above i i Fractions (20 gel Chromatography column are found by [16 to Com ml. each) are tested for b1oact1v1ty against Sarcina lutea tain antibiotic EM-2.Th'ese fractions are combined and and annblotlc composmon by 9' Results are concentrated to dryness to give a residue which is disq solved in 5 ml. of methanol, and this solution is mixed with 200 ml. of ether. The resulting precipitated mate- Fraction Zone size mam Zone Size rial is isolated by filtration; yield, 260mg. of antibiotic No. (mm) No. (mm) EM-Z preparation designated as 137.2. This preparalo 0 510 20 tion is processed further as disclosed in Part C (6). The 20 0 520 21 remaining filtrate is concentrated to dryness to give 530 20 preparation 137.3. The above obtained preparations I 5:8 28 are tested against several microorganisms on a standard 450 0 560 20 disc-plate assay (12.5 mm disc) using standard nutrient 5 238 "aces 7 8 media. All preparations are tested at concentrations of 480 20 590 20 1O mg./ml. except preparations 126.4 and 137.3. These :38 2% 8 two preparations are dissolved in 5 ml. of absolute 620 19 770 15 methanol and this solution is tested. The results of the 630 19 780 14 tests are as follows: 2:8 :3 38 :3

Prep. No. and Zone (mm) Microorganism I26 1 126.2 126.3 126.4 137.1 137.2 137.3

S. [urea 19 0 0 l4(h) 18 16.5 traces S. [urea-124 22 O 0 15(h) 22 19 traces S. aureus 21 0 0 17.5 l8 l5 l1(h) a. .ruhtilis 20 0 0 20.5 18.5 16 11(11 B. cereus 18.5 0 0 9 17.5 16.5 0 M. avium 23 l l traces 12(h) S. gallinumm l0 0 0 25.5 9 13 21 S. .rthotlmuelleri traces 10 23 P. vulgaris 11.5 0 0 30 11 15 26 E. coli traces 0 0 26 traces 9 22 K. pneumonine l l 0 0 23 traces 9 20 Note: (h) indicates a halo zone. 0 no zone outside of disc.

Fraction Zone size Fraction Zone size No. (mm) No. 660 18 810 I5 670 I7 820 15.5 680 I7 830 I5 690 17 840 I35 700 I7 850 13 710 I7 860 I3 720 16.5 870 12.5 730 16.5 880 I 740 I6 890 1 1.5 750 I6 900 10.5 760 I Fractions 470-560 contain antibiotic EM-4 only. They are combined and concentrated to dryness to give preparation 85.1.

Fractions 570-610 contain EM-4 and traces of EM-3. They are also combined and concentrated to dryness to give preparation 85.2.

Fractions 630-900 contain antibiotic EM-3 only. These fractions are combined and concentrated to dryness to give 650 gm. of a preparation of crystalline antibiotic EM-3. This crystalline preparation is dissolved in ml. of methanol under heating on a steam bath. Water (35 ml.) is added and the mixture is allowed to stand at room temperature for 4 hours. The resulting precipitated crystalline material, colorless needles, is isolated by filtration and dried; yield, 250 mg. of crystalline antibiotic EM-3.

We claim:

1. Antibiotic EM-2, which is active against various gram-positive bacteria, and which in its essentially pure crystalline form a. has the following elemental analyses: C, 57.47; H,

7.70; N, 12.99; 0, 21,87 (by difference);

b. has a melting point of 26l.4 C.;

c. has a specific rotation (01],, +5 (c, 1.0, methanol); d. has the following ultraviolet absorption spectrum:

In methanol:

A max (mg) a 216(sh) 24.02 269(sl.sh) 2.67 273 2.81 281 2.97 289 2.62;

e. is soluble in dimethylformamide, dimethylsulfoxide and lower alcohols; is less soluble in chlorinated hydrocarbon solvents and ethyl acetate; is

rather insoluble in acetone and ether; and is com-- In methanol:

A max (m#) a 243(sl.sh) 0.48 253 0.43 257 0.46 261(sh) 0.43 264 0.44 267 0.41;

e. is soluble in dimethylformamide, dimethylsulfoxide and lower alcohols; is less soluble in chlorinated hydrocarbon solvents and ethyl acetate; is rather insoluble in acetone and ether; and is completely insoluble in saturated hydrocarbon solvents; and

f. has a characteristic infrared absorption spectrum when suspended in mineral oil mull as shown in FIG. 2 of the drawing.

3. Antibiotic EM-4, which is active against various gram-positive bacteria, and which in its essentially pure crystalline form a. has'the following elemental analyses: C, 57.26; H,

7.45; N, 14.22; 0, 21.08 (by difference;

b. has a melting point of 239.8 C.; l

c. has a specific rotation M1,, +13.5 (c, 1.0,

methanol);

d. has the following ultraviolet absorption spectrum:

In methanol:

e. is soluble in dimethyformamide, dimethylsulfoxide and lower alcohols; is less soluble in chlorinated hydrocarbon solvents and ethyl acetate; is rather insoluble in acetone and ether; and is completely insoluble in saturated hydrocarbon solvents; and

f. has a characteristic infrared absorption spectrum when suspended in mineral oil mull as shown in FIG. 3 of the drawing.

4. A process for preparing the antibiotics EM-2, EM-3, and EM-4, which comprises cultivating Emericellopsis microspora strain 333 having the identifying characteristics of NRRL 5648, in an aqueous nutrient medium under aerobic conditions, to which has been added an effective amount ranging from about 1 mg./l. to about 4 gm./l. of propylproline, until substantial antibiotic activity is imparted to said medium by the production of antibiotics EM-2, EM-3 and EM-4.

5. A process, according to claim 4, wherein said aqueous nutrient medium contains a source of assimilable-carbohydrate and assimilable nitrogen.

6. A process, according to claim 4, wherein an effective amount of 4(R)-propyl-L-proline is added to the fermentation medium.

7. A process f a ccording to claim 5, wherein '4(R)-propyl-L-proline is added to the fermentation medium inthe amount of mg./l0 ml. of said medium.

8. A process for preparing the antibiotics EM-Z, EM-3 and EM-4, which comprises cultivating Emericellopsis microspora strain 333 having the identifying characteristics of NRRL 5648, in an aqueous nutrient medium under aerobic conditions, to which has been added an st vs am u ranging, fwm P9st.!..!!Ls-Z!,-.

to about 4 of P PY P until substantial anti- 

2. Antibiotic EM-3, which is active against various gram-positive bacteria, and which in its essentially pure crystalline form a. has the following elemental analyses: C, 58.46; H, 7.78; N, 13.27; O, 21.49 (by difference); b. has a melting point of 256.9* C.; c. has a specific rotation ( Alpha )D25 +12* (c, 1.0, methanol); d. has the following ultraviolet absorption spectrum; In methanol:
 3. Antibiotic EM-4, which is active against various gram-positive bacteria, and which in its essentially pure crystalline form a. has the following elemental analyses: C, 57.26; H, 7.45; N, 14.22; O, 21.08 (by difference; b. has a melting point of 239.8* C.; c. has a specific rotation ( Alpha )D25 + 13.5* (c, 1.0, methanol); d. has the following ultraviolet absorption spectruM: In methanol:
 4. A process for preparing the antibiotics EM-2, EM-3, and EM-4, which comprises cultivating Emericellopsis microspora strain 333 having the identifying characteristics of NRRL 5648, in an aqueous nutrient medium under aerobic conditions, to which has been added an effective amount ranging from about 1 mg./l. to about 4 gm./l. of propylproline, until substantial antibiotic activity is imparted to said medium by the production of antibiotics EM-2, EM-3 and EM-4.
 5. A process, according to claim 4, wherein said aqueous nutrient medium contains a source of assimilable carbohydrate and assimilable nitrogen.
 6. A process, according to claim 4, wherein an effective amount of 4(R)-propyl-L-proline is added to the fermentation medium.
 7. A process, according to claim 6, wherein 4(R)-propyl-L-proline is added to the fermentation medium in the amount of 5 mg./10 ml. of said medium.
 8. A process for preparing the antibiotics EM-2, EM-3 and EM-4, which comprises cultivating Emericellopsis microspora strain 333 having the identifying characteristics of NRRL 5648, in an aqueous nutrient medium under aerobic conditions, to which has been added an effective amount ranging from about 1 mg./l. to about 4 gm./l. of propylproline, until substantial antibiotic activity is imparted to said medium by the production of antibiotics EM-2, EM-3 and EM-4, and isolating said antibiotics EM-2, EM-3 and EM-4 as a mixture from the fermentation broth.
 9. A process, wherein a fermentation mixture of antibiotics EM-2, EM-3 and EM-4, as described in claim 8, are separated into their individual entities by silica gel chromatography. 